The present invention relates to gradient high performance liquid chromatography using a splitted tubing apparatus in which in order to achieve gradient elution, a mobile phase supplied by a pump is split into plural passages so as to be joined again thereafter.
A low flow high performance liquid chromatography (HPLC) apparatus using a micro column having an inner diameter of less than 0.1 mm, a semi-micro column or a capillary column is suitable for high-sensitive analysis of trace components, and in particular an HPLC/ESI-MS system connected online to an electro-spray ionization mass spectrometer (ESI-MS) is widely used in various fields. Recently, there have been advances to increase the sensitivity of a spray probe of the ESI-MS, so that an example is reported, in which the measurement at flow velocity from several tens of nano-liters/minute to 1 xcexcL/min becomes practical (M. Wilm, M. Mann, Anal. Chem., 68 (1996) 1.). Following this, trends toward micronization in the HPLC system are also accelerated, so that a low flow high performance liquid chromatographic system corresponding to the most suitable flow velocity of the ESI is under development. However, it is difficult to achieve a high reproducibility at flow speeds of less than several micro-liters/minute in a low high performance liquid chromatography, so that even in an available micro HPLC system with the highest performance, it is reported that the flow velocity at which the high reproducibility of gradient elution can be obtained is 10-20 xcexcL/min or more (E. C. Huang, J. D. Henion; Anal. Chem., 63 (1991) 732; A. Ducret, N. Bartone, P. A. Haynes, A. Blanchard, R. Aebersold, AnaL. Biochem., 265 (1998) 129.).
Currently, in order to perform gradient elution at a flow velocity of several micro-liters/minute or less, there are roughly two classes of methods known. These are a pre-column-flow-split method in which the gradient elution is performed at a medium-high flow velocity at which the reproducibility is obtained and the required amount of flow is split therefrom to be used (W. J. Henzel, J. H. Bourell, J. T. Stults, AnaL. Biochem., 187 (1990) 228.; E. C. Huang, J. D. Henion, Anal. Chem., 63 (1991) 732; J. P. Chervet, M. Ursem, J. P. Salzmann, Anal. Chem., 68 (1996) 1507.), and a one-chamber gradient method using one pump and one solvent mixing device (T. Takeuchi, D. Ishii, J. Chromatogr., 253 (1982) 41; J. E. Macnair, G. J. Opiteck, J. W. Jorgenson, M. A. Moseley III, Rapid Commun. Mass Spec. 11 (1997) 1279; A. Ducret, N. Bartone, P. A. Haynes, A. Blanchard, R. Aebersold, Anal. Biochem., 265 (1998) 129)
There are, however, major defects in both the methods. That is, in the pre-column-flow-split method, because a split ratio changes depending on the viscosity of effluent, the precise adjustment of the gradient flow is difficult; the one-chamber gradient method is limited basically to a gradient with an exponentially functional curve, although the instrument thereof is extremely simple and the reproducibility is high.
In a gradient high performance liquid chromatography, in order to analyze and evaluate ultra-micro-organic-components or ultra-micro-impurities in environmental samples or in medicines and the like, there is an extreme demand for the development of an apparatus in which an arbitrary gradient pattern having excellent reproducibility can be readily achieved.